Eukaryotic nitrate reductase (NaR), which catalyzes the pyridine nucleotide-dependent reduction of nitrate to nitrite, exists in nature in three forms: NADH: NaR (EC 1.7.1.1, formerly EC 1.6.6.1); NAD(P)H: NaR (EC 1.7.1.2, formerly EC 1.6.6.2); and NADPH: NaR (EC 1.7.1.3, formerly EC 1.6.6.3). These three forms of NaR are very similar in molecular composition, with the holo-enzyme containing one equivalent each of flavin adenine dinucleotide, heme-iron, and molybdenum-molybdopterin bound to an approximately 100,000 Dalton polypeptide subunit, which dimerizes to constitute the catalytically active enzyme (Redinbaugh and Campbell, 1985; Solomonson and Barber, 1990; Campbell, 1999; 2001).
Natural holo-NaR has been purified and utilized from yeasts and fungi (Fischer et al., 1992, U.S. Pat. No. 5,169,758; Johannssen et al., 1994, U.S. Pat. No. 5,294,539). Eukaryotic holo-NaR was demonstrated to work as a component of a nitrate removal bioreactor for remediation of potable water when formulated with electron-carrying dyes and bacterial denitrification enzymes (Mellor et al., 1992; U.S. Pat. No. 5,403,450). NaR has also been formulated in various types of nitrate biosensors for the detection of nitrate in water where the enzyme was “wired” to an electrode by mediating electron transfer with electron-carrying dyes, such as methyl viologen, or other electron-carrying complexes mediating electron transfer between the enzyme and the electrode of the biosensor (Glazier et al, 1998; Campbell, 1999). U.S. Pat. Nos. 5,942,388; 5,942,103; and 5,922,616). Although holo-NaR performs well in these applications, it would be desirable to produce a simplified NaR system that retains the useful nitrate-reducing properties of holo-NaR, but is smaller and less complex in structure.
Although the complete three-dimensional structure of NaR is not known, the binding sites for the 3 internally bound cofactors (namely, flavin adenine dinucleotide, heme-iron, and molybdenum-molybdopterin) have been assigned to different parts of the NaR polypeptide sequence. In the carboxyl-terminal region of the NaR polypeptide, flavin adenine nucleotide cofactor binds and the polypeptide folds to generate the pyridine nucleotide active site where electrons are donated to the enzyme by NADH or NADPH or both depending on the type of NaR. Thus, it is this region of the enzyme where variation in structure is found for the three forms of NaR which exist in nature (Shiraishi et al., 1998). In the middle of the NaR polypeptide, the heme-iron cofactor binds to generate a cytochrome b type structure, which acts as an intermediate in catalysis to transfer electrons from the reduced flavin adenine dinucleotide to the nitrate-reducing active site. In the amino-terminal region of the NaR polypeptide, the molybdenum-molybdopterin cofactor binds and the polypeptide folds into a complex shape which constitutes the nitrate-reducing active site. Also in this amino-terminal region of the NaR polypeptide is a portion which constitutes the interface for stabilizing the dimeric structure of the enzyme that is required for catalytic activity. Thus, it appears that the dimer interface structure contributes to the formation of the nitrate-reducing active site where the molybdenum-molybdopterin is bound to the NaR polypeptide via a specific cysteine residue and other amino acid residues nearby in the molybdenum-molybdopterin binding region of the NaR polypeptide (Campbell, 1999; 2001).
The nitrate-reducing molybdenum-molybdopterin-containing amino-terminal fragment of NaR has not previously been produced in any recombinant expression system. However, other fragments of Na-R have been produced recombinantly. For example, the flavin adenine nucleotide-containing carboxyl-terminal fragment of holo-NaR, which is known as the cytochrome b reductase fragment of NaR, has been expressed in recombinant form in Escherichia coli and studied by site-directed mutagenesis of active site residues and pyridine nucleotide binding site residues (Hyde and Campbell, 1990; Dwivedi et al., 1994; Shiraishi et al., 1998). This carboxyl-terminal fragment of NaR catalyzes NADH-dependent ferricyanide reduction. Its three-dimensional structure has been determined by x-ray diffraction analysis (Lu et al., 1994). Likewise, the combined fragment containing both the heme-iron-containing cytochrome b structure and the flavin adenine nucleotide-containing fragment in a single polypeptide, which is called the molybdenum reductase fragment of NaR and catalyzes NAD(P)H-dependent mammalian cytochrome c reductase activity, has been recombinantly expressed in E. coli and Pichia pastoris (Campbell, 1992; Mertens, 1999; Mertens et al., 2000). Although the structure of the molybdenum reductase fragment has not been determined, a three-dimensional model was generated by combining the structures of mammalian cytochrome b and the flavin adenine nucleotide-containing cytochrome b reductase fragment of NaR (Lu et al., 1995).
The functionality for enzymatic nitrate reduction of the nitrate-reducing molybdenum-molybdopterin-containing amino-terminal fragment of NaR was previously demonstrated by mild proteolytic cleavage of the holo-NaR from Chlorella vulgaris, which yielded a purified enzyme fragment containing the cytochrome b domain in combination with the nitrate-reducing molybdenum-molybdopterin-containing amino-terminal fragment of NaR (Solomonson and Barber, 1990).
Recombinant catalytically-active holo-NaR has been expressed in P. pastoris and some other systems including plants and fungi (Su et al., 1996; Su et al., 1997; Mertens, 1999; George et al., 1999; Campbell, 1999; 2001; Skipper et al., 2001). Thus, it is clear that the methylotrophic yeast, P. pastoris, is capable of producing a recombinant form of the complete NaR polypeptide as well as the three cofactors required for formation of the active enzyme. Furthermore, P. pastoris has recently been shown to produce Pichia angusta (formerly known as Hansenula polymorpha) NAD(P)H: NaR (YNaR1; EC 1.7.1.2, formerly EC 1.6.6.2) (Barbier and Campbell, 2000), which was cloned from this yeast that is closely related to P. pastoris (Avila et al., 1995).
Methylotrophic yeasts, such as P. pastoris, offer many advantages over bacteria for production of eukaryotic proteins, which include the ability to produce complex proteins like NaR and its catalytically active fragments (Su et al., 1997; Mertens et al., 2000). The P. pastoris expression system, which has been described previously (see, for example, U.S. Pat. Nos. 5,166,329; 5,122,465; 5,032,516; 5,004,688; 5,002,876; 4,929,555; 4,895,800; 4,885,242; 4,882,279; 4,879,231; 4,857,467; 4,855,231; 4,837,148; 4,818,700; 4,812,405; 4,808,537; and 4,683,293, the disclosures of which are hereby incorporated by reference), has been used to produce a number of recombinant proteins including the pharmaceutical insulin-like growth factor-1 (Brierley et al. 1994; U.S. Pat. No. 5,324,639), pertactin antigen (Clare et al., 2001; U.S. Pat. No. 6,197,548) and enzymes (Payne et al., 1998). In some cases, the protein of interest is produced by secretion into the media as was done for pharmaceutical insulin-like growth factor-1. However, NaR is not secreted into the media, but rather, it is produced intracellularly as a soluble protein in the cytoplasm of the methylotrophic yeast cells (Su et al., 1997; Barbier and Campbell, 2000; Skipper et al., 2001). This requires the yeast to be extracted to obtain the soluble proteins and the NaR must be purified from the other soluble proteins to obtain the purified enzyme.